Nearly 50 million Americans experience severe or chronic pain, costing about 60 billion dollars annually in lost productivity. About 22-26% of the general population of the United States state that the pain they experience is orofacial in nature, including toothaches, temporomandibular joint pain, and facial pain. Orofacial pain is generally ranked to be more severe than peripheral pain, partly due to dense innervation of orofacial structures. We have identified the enzyme cyclin dependent kinase 5 (Cdk5) as having a role in pain signaling. The enzymatic activity of Cdk5 is upregulated downstream of inflammation, which is pertinent to orofacial pain as most is caused inflammation. Even though Cdk5 activity is intriguingly upregulated following inflammation, we also needed to determine if this activity change, in turn, had an actual impact on pain processing. We therefore tested genetically engineered mice with either have increased or decreased Cdk5 activity for their reactions to painful stimuli. We employed traditional pain testing that is reflex based (such as paw withdrawal) to examine the responses of the mice but have also developed operant assays that essentially uses a conflict/reward paradigm to allow the animals to make a choice whether to withdraw from an aversive stimulus or endure pain to achieve a reward. These behavioral tests additionally provide investigator-independent measurement of pain using automatically recorded behavior of the observed animals, so they incur less stress, and their behavior can be measured repeatedly in a non-biased fashion. Currently, our lab has three behavioral testing devices to measure mechanical, thermal, and chemical related pain in the orofacial region. Mechanical pain is measured using an Orofacial Stimulation Test (OST) that is designed so that the mice must stick their snout through a drinking window that can be equipped with abrasive wires to get access to the reward bottle containing 30% sucrose. Using a similar device to OST called OPAD (Orofacial Pain Assessment Device), we have also examined the effects of Cdk5 on thermal sensitivity, where a mouse must make contact with two thermodes to access the reward bottle. The thermodes can be set at noxious hot or cold temperatures. Lastly, chemical aversion can be tested using a device called the lickometer to measure the consumption of water spiked with offensive chemicals, including the pungent plant compounds capsaicin and mustard oil. Our behavioral testing has basically shown that increased Cdk5 activity leads to hyperalgesia (increased sensitivity to painful stimuli) while decreased activity causes hypoalgesia (decreased sensitivity to pain). A means to regulate Cdk5 activity in mice, as mentioned above, is by genetically manipulating the levels of its regulatory subunit p35 (Cdk5r1). Genetic deletion of p35 causes up to a 90% reduction in Cdk5 activity while overexpression produces a 50% increase, resulting in hypoalgesia or hyperalgesia respectively. While there is another known regulatory subunit p39 (Cdk5r2) that accounts for the residual Cdk5 activity following deletion of p35, our lab has determined that the activator p39 plays no role in regulating pain signaling. So, with this data, we are now focused solely on studying Cdk5/p35 contributions to pain. Our lab has shown that the expression of p35 is the rate limiting step for Cdk5 activity and the key regulatory subunit affecting Cdk5 activity during pain states. Therefore, we plan monitor p35 expression not only in future animal models of pain but also in samples from patients experiencing chronic pain. We additionally plan to focus on disrupting Cdk5/p35 interactions as a means to inhibit pain hypersensitivity. Unlike senses like vision and smell, a major component of nociception, the detection of noxious stimuli, involves the activation of ion channels on the peripheral nerve ending, resulting in depolarization of the neuron and subsequent neuronal firing. During inflammation, ion channels that relay painful mechanical, thermal, or chemical stimuli can become sensitized and display altered channel activity that promotes hyperalgesia. We have shown that Cdk5 activity is induced under inflammation and, this, in turn, has effects on pain sensitivity. We are currently trying to identify substrates of Cdk5 that are involved in pain perception. Previously, we identified transient receptor potential vanilloid receptor 1 (TRPV1), an ion channel that is activated by noxious heat and acidity, as a substrate of Cdk5. We have recently identified two other nociceptive ion channels as being substrates of Cdk5. The ion channel transient receptor potential ankyrin 1 (TRPA1) is often coexpressed in the same pain sensing neurons as TRPV1 and is known to act as a chemosensor needed for the detection of endogenous noxious compounds, plant irritants like mustard oil, and environmental toxins. Our lab showed that Cdk5 modifies TRPA1 causing more cultured neurons to respond to mustard oil and greater in vivo oral aversion to mustard oil in mice. An additional gated ion channel substrate of Cdk5 is the Purinergic Receptor P2X (P2X2a) which is involved in the detection of chemical signals released upon tissue injury. We have shown that the P2X2a channel is more responsive to use over time than normal when modified by Cdk5. In summary, having shown that Cdk5 modulates orofacial mechanical pain, our current research is focused on further confirming these findings with additional Cdk5 mouse models. Furthermore, we will vigorously pursue molecular investigations into identifying novel Cdk5 substrates involved in pain signaling. Additionally, we will continue our efforts to collaboratively study role of TGF-beta in disease processes affecting orofacial tissues.